*/Disease Information (sort of like the Intro to your Mini Research Write up): Trypanosoma brucei is a parasite that causes African Trypanosoma, which is also known as the "sleeping sickness." Tsetse flies, which are only found in rural Africa, transmit the parasite through a bite. Furthermore, the disease can be transmitted via mother to child, sexual contact, or other blood sucking inseects. Annually, 10,000 new cases of the African Trypanosoma are reported to the World Health Organization (WHO). African Trypanosoma is curable when treated but is fatal when untreated. There are speculations that many cases of the "sleeping sickness" are undiagnosed and therefore untreated.Treatments include Pentamidine, Suramin, Melarsoprol, Eflornithine, and Nifurtimox. Human African Trypanosomiasis takes 2 forms: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Humans infected with Trypanosoma brucei gambiense can take months or years before developing the first symptoms while humans infected with Trypanosoma brucei rhodesiense can take few weeks or months before developing the first symptoms. Animals can host the human pathogen parasites, making the disease more widespread.
Essentiality of this protein: Adenylate kinase is essential for several reasons. For instance, without this protein, it causes embryonic lethal/arrest. Many research has been done to understand the essentiality of adenylate kinase. This protein is involved in energetic metabolism in the liver of rats. When rats are infected by Trypanosoma evansi, the function of adenylate kinase is hindered, and leads to liver injury and inflammatory infiltration. Therefore, adenylate kinase is an essential protein. https://www.ncbi.nlm.nih.gov/pubmed/26239575
Structure (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: None -- For Homology Model option: N/A ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
Figure 3. The BLASTP search NCBI against the PDB for Adenylate kinase found in Trypanosomabrucei ---- Query Coverage: 67% ---- Max % Identities: 43% ---- % Positives: N/A ---- Chain used for homology: 1ZAK A
Expression Information (has it been expressed in bacterial cells): Adenylate kinase in Trypanosomabruceihas not been expressed before. http://www.tdrtargets.org/targets/view?gene_id=15709 (Under Expression heading)
Purification Method: There is no purification since there is no expression
Image of protein (PyMol with features delineated and shown separately): No PDB structure available
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MAVLSEDVLLYLKEKNIPMLFEQIVQNIISDAPERPMSYIGDLMRRGIPLQIFIAGPAGS
GKRTQCKNIADRLGVVLISSGQVLTRGVESGSETSQLAHSYVSRGERVPDTLVSMIMKDR
LSQSDACEKGWLVEGYPRNAQQAQAVEECGVIPQVFILLDLPEDLSFRRLEHRRYDPATN
KXYHMLDNPPPGGRCRVMRTAPAEGCKFP
*length of your protein in Amino Acids 212
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website 23283.05
Molar Extinction coefficient of your protein at 280 nm wavelength: 14690
*CDS Gene Sequence (paste as text only): atggcggtgctgagcgaagatgtgctgctgtatctgaaagaaaaaaacattccgatgctg tttgaacagattgtgcagaacattattagcgatgcgccggaacgcccgatgagctatatt ggcgatctgatgcgccgcggcattccgctgcagatttttattgcgggcccggcgggcagc ggcaaacgcacccagtgcaaaaacattgcggatcgcctgggcgtggtgctgattagcagc ggccaggtgctgacccgcggcgtggaaagcggcagcgaaaccagccagctggcgcatagc tatgtgagccgcggcgaacgcgtgccggataccctggtgagcatgattatgaaagatcgc ctgagccagagcgatgcgtgcgaaaaaggctggctggtggaaggctatccgcgcaacgcg cagcaggcgcaggcggtggaagaatgcggcgtgattccgcaggtgtttattctgctggat ctgccggaagatctgagctttcgccgcctggaacatcgccgctatgatccggcgaccaac aaannntatcatatgctggataacccgccgccgggcggccgctgccgcgtgatgcgcacc gcgccggcggaaggctgcaaatttccg
*GC% Content for gene: 59.5%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): N/A (according to Diane) *GC% Content for gene (codon optimized): N/A (according to Diane)
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
*NCBI Gene # or RefSeq#: 3663067
*Protein ID (NP or XP #) or Wolbachia#: XP_009315768.1
*Organism (including strain): Trypanosoma brucei
Etiologic Risk Group (see link below): RG2
https://my.absa.org/tiki-index.php?page=Riskgroups
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Trypanosoma brucei is a parasite that causes African Trypanosoma, which is also known as the "sleeping sickness." Tsetse flies, which are only found in rural Africa, transmit the parasite through a bite. Furthermore, the disease can be transmitted via mother to child, sexual contact, or other blood sucking inseects. Annually, 10,000 new cases of the African Trypanosoma are reported to the World Health Organization (WHO). African Trypanosoma is curable when treated but is fatal when untreated. There are speculations that many cases of the "sleeping sickness" are undiagnosed and therefore untreated.Treatments include Pentamidine, Suramin, Melarsoprol, Eflornithine, and Nifurtimox. Human African Trypanosomiasis takes 2 forms: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Humans infected with Trypanosoma brucei gambiense can take months or years before developing the first symptoms while humans infected with Trypanosoma brucei rhodesiense can take few weeks or months before developing the first symptoms. Animals can host the human pathogen parasites, making the disease more widespread.
Link to TDR Targets page (if present): N/A
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
https://www.ncbi.nlm.nih.gov/gene/3663067
Essentiality of this protein:
Adenylate kinase is essential for several reasons. For instance, without this protein, it causes embryonic lethal/arrest. Many research has been done to understand the essentiality of adenylate kinase. This protein is involved in energetic metabolism in the liver of rats. When rats are infected by Trypanosoma evansi, the function of adenylate kinase is hindered, and leads to liver injury and inflammatory infiltration. Therefore, adenylate kinase is an essential protein.
https://www.ncbi.nlm.nih.gov/pubmed/26239575
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Monomer
Complex of proteins?: Not complex
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
http://pubs.acs.org/doi/pdf/10.1021/pr500813f
*EC#: 2.7.4.3
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=2.7.4.3
-- Show screenshot of BRENDA enzyme mechanism schematic
Figure 1. BRENDA enzyme mechanism schematic of the Adenylate kinase in Trypanosoma brucei
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Continuous Spectrophotometric Rate Determination
-- link to Sigma (or other company) page for assay (see Sigma links below) or link (or citation) to paper that contains assay information
http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-myokinase.html
-- links to assay reagents (substrates) pages.
http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-myokinase.html
--- List cost and quantity of substrate reagents, supplier, and catalog #
Figure 2. Cost and quantity of substrate reagents, supplier, and catalog number of Adenylate kinase
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: None
-- For Homology Model option: N/A
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
Figure 3. The BLASTP search NCBI against the PDB for Adenylate kinase found in Trypanosoma brucei
---- Query Coverage: 67%
---- Max % Identities: 43%
---- % Positives: N/A
---- Chain used for homology: 1ZAK A
Current Inhibitors: α, w-dinucleoside 5'-polyphosphates
http://onlinelibrary.wiley.com/store/10.1111/j.1432-1033.1975.tb02291.x/asset/j.1432-1033.1975.tb02291.x.pdf;jsessionid=D09085B9C78C2B0CD26D4B25C6C1CE0A.f01t03?v=1&t=j2draat4&s=afbda48816196eb499054be191c8ef98dd1b77b9
Expression Information (has it been expressed in bacterial cells): Adenylate kinase in Trypanosoma brucei has not been expressed before.
http://www.tdrtargets.org/targets/view?gene_id=15709 (Under Expression heading)
Purification Method: There is no purification since there is no expression
Image of protein (PyMol with features delineated and shown separately): No PDB structure available
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MAVLSEDVLLYLKEKNIPMLFEQIVQNIISDAPERPMSYIGDLMRRGIPLQIFIAGPAGS
GKRTQCKNIADRLGVVLISSGQVLTRGVESGSETSQLAHSYVSRGERVPDTLVSMIMKDR
LSQSDACEKGWLVEGYPRNAQQAQAVEECGVIPQVFILLDLPEDLSFRRLEHRRYDPATN
KXYHMLDNPPPGGRCRVMRTAPAEGCKFP
*length of your protein in Amino Acids 212
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website 23283.05
Molar Extinction coefficient of your protein at 280 nm wavelength: 14690
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Figure 4. TMpred output graph for Adenylate kinase found in Trypanosoma brucei
*CDS Gene Sequence (paste as text only):
atggcggtgctgagcgaagatgtgctgctgtatctgaaagaaaaaaacattccgatgctg tttgaacagattgtgcagaacattattagcgatgcgccggaacgcccgatgagctatatt ggcgatctgatgcgccgcggcattccgctgcagatttttattgcgggcccggcgggcagc ggcaaacgcacccagtgcaaaaacattgcggatcgcctgggcgtggtgctgattagcagc ggccaggtgctgacccgcggcgtggaaagcggcagcgaaaccagccagctggcgcatagc tatgtgagccgcggcgaacgcgtgccggataccctggtgagcatgattatgaaagatcgc ctgagccagagcgatgcgtgcgaaaaaggctggctggtggaaggctatccgcgcaacgcg cagcaggcgcaggcggtggaagaatgcggcgtgattccgcaggtgtttattctgctggat ctgccggaagatctgagctttcgccgcctggaacatcgccgctatgatccggcgaccaac aaannntatcatatgctggataacccgccgccgggcggccgctgccgcgtgatgcgcacc
gcgccggcggaaggctgcaaatttccg
*GC% Content for gene: 59.5%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): N/A (according to Diane)
*GC% Content for gene (codon optimized): N/A (according to Diane)
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**